This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

NOTA: O seguinte protocolo está de acordo com os Institutos Nacionais de Saúde como diretrizes aprovados pela Universidade de Oregon Animal Care e do Comitê Use. 1. Cirurgia aguda <ol><li> Anestesiar o animal com um cocktail de cetamina-medetomidina, via intraperitoneal (ip) (Tabela 1). NOTA: Os ratos usados ​​nestas experiências são gerados pelo cruzamento de um ChR2-EYFP line10 transgénico dependente de cre a Interneuron linhas de driver (Pvalb-iCre11, PV +; Sst-iCre12, SOM +; Cr-iCre12, CR +). Entrega Viral de ChR2 ou opsinas relacionados devem funcionar igualmente bem, assumindo níveis de expressão semelhantes são obtidos. </li><li> Antes de iniciar a cirurgia, garantir que as exposições de animais não resposta a uma pitada toe suave. Re-administração de anestésicos durante todo o experimento como necessário para manter este profundidade da anestesia. Se usar anestésicos injetáveis, opcionalmente implantar um cateter ip para injeções de manutenção. </li><li> Manter o animal hidratado com solução de Ringer com lactato ao longo da experiência (aproximadamente 3 ml / kg / h), por exemplo, utilizando um cocktail de anestésico apropriadamente diluído para injecções de manutenção (Tabela 1) ou solução salina. </li><li> Colocar o animal em um aparelho de cabeça-holding estereotáxica ou outro. Certifique-se que o crânio é bem protegido. Isto é essencial para a manutenção de gravações de célula única estáveis. </li><li> Aplicar pomada opthalamic aos olhos para evitar o ressecamento. Manter a temperatura corporal a 36,5-37 ° C. </li><li> Realize um dreno cisternal para a estabilidade de gravação adicional. Usando uma lâmina de bisturi, remover o tecido a partir da face posterior do crânio para expor a cisterna magna. Faça um pequeno corte na dura para drenar o líquido cefalorraquidiano. Use somente a ponta da lâmina, e evitar entrar em contato com a haste cerebelo ou cérebro. </li><li> Usando o bisturi, realizar uma craniotomia pequena (~ 2 milímetros 2) sobre a área cortical de interesse (por auditivocórtex, cerca de -2,3 mm posterior do bregma, 4.5 mm lateral da linha média). </li><li> Remover a dura-máter e cobrir o córtex exposto, com uma camada de agarose a quente 0,5-1 mm de espessura (1,5% de agarose em solução salina, 0,9% de NaCl; aplicam-se a ~ 37 ° C). Manter a húmido de agarose ao longo da experiência pela aplicação periódica de várias gotas de solução salina. </li></ol> 2. Gravação de Set-up <ol><li> Prepare um eletrodo de tungstênio (14/07 mohms, 127 mm de diâmetro, 12 ° ponta cônica, epóxi-revestido). Supercola o eletrodo a um tubo capilar de vidro e adicione psiquiatra tubulação de calor para grip (Figura 1). </li><li> Monte o eletrodo em um micromanipulator motorizada (ou hidráulica), definida para viajar ortogonal à superfície cortical. Deslize um fio terra sob a pele, contra o crânio. Evitar o contacto com os músculos do lado da cabeça e a parte de trás do pescoço enquanto eles podem gerar artefactos eletromiográficas. </li><li> Amplificar o sinal elétrico usando um amplificador extracelularli er fi adequado para a gravação em uma única unidade, de preferência equipado com um modo de verificação de impedância. Monitorar a atividade spiking em curso (passa-banda 300 - 5000 Hz) com dois osciloscópios e um conjunto de alto-falantes. Aqui, os dados gravados são digitalizados de forma contínua a uma taxa de amostragem de 10 kHz; picos são extraídos off-line. </li><li> Avance o eléctrodo através da agarose até que a ponta atinge a superfície do córtex. Observe este passo através de um microscópio. &quot;Zero&quot; esta posição no micromanipulator. </li><li> Para aproximar melhor a profundidade de gravação, confirmar visualmente que a ponta do eléctrodo sai da superfície cortical, a uma profundidade de zero quando a retirada do eléctrodo na extremidade de uma penetração. NOTA: A discrepância entre a leitura do manipulador e da posição do eletrodo observado indica que ele se afastou em relação ao tecido. Isto pode ser causada pela instabilidade do cérebro ou animal, ou manipulador deriva. <ol><li> Como uma verificação adicional para garantir queeste conjunto de ponto zero corresponde à superfície do córtex, monitorizar potenciais de campo evocadas por estímulo enquanto avança através das primeiras várias centenas de micrómetros de tecido. Ao monitorar potenciais de campo, diminuir o menor corte do filtro passa-banda do amplificador extracelular para 10 Hz. Confirmar que a polaridade do potencial de campo local inverte em torno de uma profundidade de ~ 100 pm, perto da fronteira I / II 13 camada. Este é um ponto de referência confiável para o córtex auditivo, mas pode variar para outras áreas corticais. </li></ol></li><li> Montar uma fibra óptica acoplada a uma fonte de luz azul (Figura 2) para um micromanipulador manual. Utilizando o microscópio, posicionar a ponta da fibra tão perto da superfície da agarose quanto possível. Centrar o feixe em que o eléctrodo vai entrar no tecido. </li><li> Regular a saída da fonte de luz com uma unidade de controle capaz de entregar um TTL (lógica transistor-transistor) trem de pulsos de largura e duratio especificadon- por exemplo, um Arduino (Figura 3), um cartão de computador de I / O (como mostrado), ou um gerador de impulsos disponíveis comercialmente. <ol><li> Monitorar o sinal em ambos os osciloscópios. </li></ol></li><li> Meça a potência total da luz (mW) na ponta da fibra óptica, utilizando um medidor de potência. Use esse valor para calcular a irradiância (mW / mm 2), dividindo pela área da secção transversal do núcleo da fibra. Comece com um valor de intensidade no intervalo de 10 - 15 mW / mm 2 e ajuste para baixo, se necessário, até artefactos são eliminados. </li><li> Verifique a existência de artefatos de luz com o eletrodo na agarose, pronta para entrar no tecido. Comece a busca de trem de pulso (por exemplo, 30 pulsos de luz ms, 500 ms intervalo interstimulus (ISI)). <ol><li> Elimine artefactos de luz transitórios (Figura 4) por reposicionamento da fibra óptica relativa ao eléctrodo para mudar o ângulo da luz incidente. Se artefatos persistir, tente diminuir o poder da luz. Em the caso raro que os artefatos não podem ser completamente eliminados (apenas minimizado), prolongar a duração do pulso de pesquisa. Isto pode ajudar a identificar os verdadeiros pontos neuronais. </li></ol></li></ol> 3. Em linha reta PINP-in &#39; <ol><li> Avance o eléctrodo lentamente através do cérebro, a uma taxa de aproximadamente 1 mm / seg. Use um osciloscópio para monitorar a atividade em curso. Por outro lado, desencadear os pulsos de laser. </li><li> Escutar para o &#39;de hash &quot;fraco de picos de luz evocado no monitor de áudio, o que indica que o eléctrodo está a aproximar de uma célula ChR2 + e pode muitas vezes ser percebido bem antes de o sinal eléctrico é aparente no osciloscópio. Diminuir a taxa de antecedência. NOTA: Se a célula situa-se no caminho do eletrodo, a atividade de luz evocada vai crescer mais (e mais alto). À medida que o eléctrodo se aproxima de uma única interneurónio, vai resolver o hash em pequenos mas bem definidas picos de tamanho e forma uniforme. </li><li> Assim que lispikes evocado-GHT são grandes o suficiente para desencadear de individualmente no osciloscópio, começar a fazer isso. Ajustar a escala no eixo horizontal para observar a forma exacta da forma de onda de pico. </li><li> Pare e espere o tecido para resolver. Resistir à tentação para mover o eléctrodo mais perto da célula. Este passo é crítico para uma leitura estável. Se depois de vários minutos a relação sinal-para-ruído não melhorou - isto é, o tecido tem resolvido, mas não trouxe a célula mais perto da ponta do eléctrodo - 5 um avanço adicional e esperar de novo. <ol><li> Repita esse processo até que o pico ou a calha do potencial de ação pode ser capturada de forma confiável com um limiar de tensão definir bem acima do ruído de fundo (por exemplo, +/- 300 mV ou mais). NOTA: Há pouco risco de resultados falso-positivos ou ambigüidade quando PINPing interneurônios inibitórios. A maioria das células pode facilmente seguir um trem de pulsos com duração de 30 ms e inter-pulintervalo de 500 ms si só, ou mais rápido, com um primeiro pico de latência de confiança de 2-5 mseg (Figura 5). A maioria das células fotovoltaicas +, em particular, pode sustentar a disparar para um total de 1 seg (Figura 6). Porque estes são neurônios inibitórios, disynaptic ativação (indireto) não é uma grande preocupação. </li></ol></li><li> Durante a gravação, monitorar a atividade em curso no primeiro osciloscópio. Mantenha um olhar atento sobre o tamanho ea forma dos picos usando o segundo osciloscópio. Observe a qualidade de seu som no alto-falante. <ol><li> Ouça com atenção para mudanças abruptas, que indicam que a célula é qualquer desenho muito próximo ao eletrodo (onde corre o risco de ser empalado ou danificado), ou está se afastando. Se os picos se tornam grandes e distorcida, voltar para fora; se tornam menores, avançar o eletrodo. Mova-se lentamente em 2 mm etapas. </li><li> Confirme a qualidade da gravação post-hoc sobrepondo todas as formas de onda de pico, alinhados ao pico ou vale. Variar os thres tensãosegurar. Através de uma ampla gama de valores de limiar, não só deve ser sempre uma forma consistente pico de altura uniforme (Figura 5a). </li></ol></li><li> Se em algum momento o sinal torna-se contaminado com pontas de um neurônio vizinho, seguir em frente. Avance lentamente, no off-chance de que o eletrodo vai passar fora do alcance da célula vizinha, mas permanecem na faixa do neurônio alvo. (Isto é geralmente sem sucesso.) </li><li> Mover o eléctrodo através de toda a profundidade do córtex (900 + im) em cada penetração. Se nenhuma luz neurônios responsivos são encontradas depois de muitas penetrações ou, pelo contrário, as gravações são rotineiramente contaminados com picos de células ChR2- vizinhos, tente um novo eletrodo. NOTA: Mesmo dentro de um único lote, a geometria de impedância e ponta de eletrodos individuais podem variar consideravelmente. Ambos os fatores contribuem para a efetiva &quot;Raio escuta&quot; do eletrodo, o que deve ser grande o suficiente para detectar picos de luz evocados while pesquisa, ainda restringido o suficiente para permitir a gravação de um único interneurónio isoladamente. </li><li> Teste a impedância de eléctrodos como desejado. Para evitar danificar o tecido, fazer isso com a ponta do eléctrodo na parte superior da camada de agarose, bem fora do cérebro. Dentro da faixa de 7-14 mohms, a impedância exata não é um preditor certeza de desempenho, ou rendimento, para esta aplicação. Dito isto, é um proxy razoável para o raio de escuta: eletrodos de baixa impedância pegar mais unidades; de alta impedância, menos. </li><li> No fim do experimento, os animais a eutanásia por meio institucional-aprovados, tais como sobredosagem anestésica, ou luxação cervical sob um plano de anestesia profunda. </li></ol>

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	<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINP:+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording.">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4, (2009).</li><li>Moore, A. K., Wehr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+inhibitory+interneurons+in+auditory+cortex+are+well-tuned+for+frequency.">Parvalbumin-expressing inhibitory interneurons in auditory cortex are well-tuned for frequency.</a> J Neurosci. 33, 13713-13723 (2013).</li><li>Merchant, H., de Lafuente, V., Pena-Ortega, F., Larriva-Sahd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+impact+of+interneuronal+inhibition+in+the+cerebral+cortex+of+behaving+animals.">Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals.</a> Prog Neurobiol. 99, 163-178 (2012).</li><li>Markram, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneurons+of+the+neocortical+inhibitory+system.">Interneurons of the neocortical inhibitory system.</a> Nat Rev Neurosci. 5, 793-807 (2004).</li><li>Atallah, B. V., Bruns, W., Carandini, M., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+interneurons+linearly+transform+cortical+responses+to+visual+stimuli.">Parvalbumin-expressing interneurons linearly transform cortical responses to visual stimuli.</a> Neuron. 73, 159-170 (2012).</li><li>Wilson, N. R., Runyan, C. A., Wang, F. L., Sur, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+and+subtraction+by+distinct+cortical+inhibitory+networks+in+vivo.">Division and subtraction by distinct cortical inhibitory networks in vivo.</a> Nature. 488, 343-348 (2012).</li><li>Letzkus, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+disinhibitory+microcircuit+for+associative+fear+learning+in+the+auditory+cortex.">A disinhibitory microcircuit for associative fear learning in the auditory cortex.</a> Nature. 480, 331-335 (2011).</li><li>Pi, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+interneurons+that+specialize+in+disinhibitory+control.">Cortical interneurons that specialize in disinhibitory control.</a> Nature. 503, 521-524 (2013).</li><li>Adesnik, H., Bruns, W., Taniguchi, H., Huang, Z. J., Scanziani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neural+circuit+for+spatial+summation+in+visual+cortex.">A neural circuit for spatial summation in visual cortex.</a> Nature. 490, 226-231 (2012).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nat Neurosci. 15, 793-802 (2012).</li><li>Hippenmeyer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+switch+in+the+response+of+DRG+neurons+to+ETS+transcription+factor+signaling.">A developmental switch in the response of DRG neurons to ETS transcription factor signaling.</a> PLoS Biol. 3, 159 (2005).</li><li>Taniguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+resource+of+Cre+driver+lines+for+genetic+targeting+of+GABAergic+neurons+in+cerebral+cortex.">A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex.</a> Neuron. 71, 995-1013 (2011).</li><li>Christianson, G. 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The authors have no competing financial interests.

Embora PINP é conceitualmente simples, ele pode ser um desafio na prática. Um dos principais determinantes do sucesso é a escolha do eletrodo. O raio de audição eléctrica é o parâmetro crítico. Ele deve ser suficientemente grande para detectar picos de luz evocado quando a ponta é ainda a certa distância a partir de uma célula ChR2 +, de modo que se pode ajustar a velocidade de avanço em conformidade. Ao mesmo tempo, deve ser restringido o suficiente para permitir um bom isolamento única unidade. Ou seja, ...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

<table border="0" cellpadding="0" cellspacing="0" width="1482">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Name of Material/Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:341px;">Product/Stock Number</td>
			<td style="width:708px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChR2-EYFP Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">12569</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pvalb-iCre (PV) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">8069</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sst-iCre (SOM) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">13044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cr-iCre (CR) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td>10774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Agarose</td>
			<td style="width:171px;">Sigma-Aldrich</td>
			<td style="width:341px;">A9793</td>
			<td>Type III-A, High EEO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Micro Point (dural hook)</td>
			<td style="width:171px;">FST</td>
			<td style="width:341px;">10066-15</td>
			<td style="width:708px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Surgical Scissors</td>
			<td style="width:171px;">FST</td>
			<td>14084-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Scalpel</td>
			<td style="width:171px;">FST</td>
			<td>10003-12 (handle), 10011-00 (blades)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Puralube Ophthalmic Ointment</td>
			<td style="width:171px;">Foster &#38; Smith</td>
			<td style="width:341px;">9N-76855</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Homeothermic Blanket</td>
			<td style="width:171px;">Harvard Apparatus</td>
			<td style="width:341px;"><a name="RANGE!C11">507220F</a></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tungsten Microelectrodes</td>
			<td style="width:171px;">A-M Systems</td>
			<td>577200</td>
			<td>12 M&#937; AC resistance, 127 &#956;m diameter, 12&#176; tapered tip, epoxy-coated</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Capillary Glass Tubing</td>
			<td style="width:171px;">Warner Instruments</td>
			<td style="width:341px;">G150TF-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Heat Shrink Tubing</td>
			<td style="width:171px;">DigiKey</td>
			<td>A332B-4-ND</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Zapit Accelerator</td>
			<td style="width:171px;">DVA</td>
			<td style="width:341px;">SKU ZA/ZAA</td>
			<td>Use with standard Super Glue.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Microelectrode AC Amplifier 1800</td>
			<td style="width:171px;">AM Systems</td>
			<td style="width:341px;">700000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">MP-285 Motorized Micromanipulator</td>
			<td style="width:171px;">Sutter</td>
			<td style="width:341px;">MP-285</td>
			<td></td>
		</tr>
		<tr height="20">
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a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

Um grande desafio em neurofisiologia tem sido a de caracterizar as propriedades de resposta e função dos inúmeros tipos de células inibitória no córtex cerebral. Nós aqui partilhar a nossa estratégia para a obtenção estáveis, bem isoladas gravações de uma única unidade de interneurônios inibitórios identificadas no córtex anestesiado mouse usando um método desenvolvido por Lima e colaboradores um. As gravações são realizadas em ratinhos que expressam channelrodopsina-2 (ChR2) em subpopulações neuronais específicas. Os membros da população são identificados pela sua resposta a um breve flash de luz azul. Esta técnica - chamada de &quot;PINP&quot;, ou Identificação fotoestimulação-assistida de Populações Neuronais - pode ser implementado com equipamento de gravação extracelular padrão. Ela pode servir como uma alternativa barata e acessível a imagiologia de cálcio ou patching visualmente-guiado, com o objectivo de direccionamento gravações extracelulares de células identificadas geneticamente. Here nós fornecemos um conjunto de diretrizes para a otimização do método na prática diária. Nós refinado nossa estratégia especificamente para alvejar as células parvalbumina-positivo (PV +), mas descobriram que ele funciona para outros tipos interneuron, bem como, tais como (CR +) interneurons expressando Calretinin-somatostatina expressando (SOM +) e.

Nós aqui partilhar a nossa estratégia para a obtenção de gravações de uma única unidade de interneurônios inibitórios geneticamente classificados no córtex anestesiado rato, utilizando um método optogenetic desenvolvido por Lima et al. A Tabela 1 detalha o cocktail anestésico sugerido, quetamina-medetomidina-acepromazina (1 &quot;. KMA &quot;). A Figura 1 representa um microeléctrodo de tungsténio, preparado para a gravação. A figura 2</strong...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Um Guia para<em&gt; In vivo</em&gt; Gravação única unidade de Optogenetically Identificado Cortical Inibitória Interneurônios

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

a-guide-to-vivo-single-unit-recording-from-optogenetically-identified

Research

JoVE Journal

Neuroscience

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

19.0K visualizações.  University of Oregon. Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching. 

Vídeo: A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Única unidade de In vivo Gravações do quiasma óptico de rato

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Neurociência

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Gravação de respostas comportamentais à Reflexão em Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

Implante cirúrgico de crônicas Eletrodos Neurais para registrar a atividade única Unidade e Sinais eletrocorticográfica

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

De gravação simultânea de sinais de cálcio dos neurônios identificados e comportamento alimentar de Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Registro da atividade elétrica dos neurônios identificados no cérebro intacto de peixes transgénicos

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracelular de gravação, Sensorial Campo Mapping, e cultivando neurônios identificados nos Sanguessuga, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

Subtipo seletivo Eletroporação de Cortical Interneurônios

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell Recordings patch-clamp de Morphologically- e identificados Neuroquimicamente hipocampo Interneurônios

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Gravação simultânea eletrofisiológica e micro-injeções de agentes inibidores no cérebro do roedor

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...